The proposed studies are directed at understanding the molecular mechanisms underlying the synthesis of nitric oxide (NO) by vascular endothelial cells. The endothelium plays a crucial role in the regulation of vascular smooth muscle tone. NO is labile molecule synthesized in and released by endothelial cells, and promotes the relaxation of vascular smooth muscle. Clinically important organic nitrate vasodilator drugs form NO in vivo, and endothelium-derived NO thus represents the "endogenous nitrovasodilator". Endothelium-dependent vascular regulation is altered in atherosclerosis, diabetes, hypertension, and septic shock. Studies of the synthesis and regulation of nitric oxide in endothelial cells are hampered by the facts that the enzyme is present in limited quantities, and the cDNA has not been isolated. Several interdependent lines of investigation are being pursued. We have determined the amino acid sequence of peptides purified from the bovine brain nitric oxide synthetase, and used these sequence data in molecular cloning experiments to isolate a cDNA clone for the bovine aortic endothelial cell nitric oxide synthetase and to explore the existence of related bovine genes. In the proposed studies, we will identify and characterize a full-length cDNA clone and identify related cDNAs in endothelial cells, and also characterize related genes. We have shown that the partially purified brain nitric oxide synthetase catalyzes the formation of stable, biologically active protein S-nitrosothiol compounds. A heterologous expression system with the full-length endothelial cell cDNA will be established, and used to characterize the chemical nature and biological activity of the nitric oxide synthetase product adducts. The activity of nitric oxide synthetase in endothelial cells is dynamically regulated by cytokines, and this may be relevant to the pathogenesis of septic shock. We will explore the molecular mechanisms whereby specific cytokines regulate nitric oxide synthetase activity. In the course of this work, antibodies to the endothelial nitric oxide synthetase will be produced. The genomic organization of the endothelial NO synthetase gene will be determined, and its cis-acting regulatory elements will be identified and characterized. The proposed research will develop biological reagents for studying the structural and regulatory features of the endothelial cell nitric oxide synthetase system, and will apply these findings to gain insight into disease states in which endothelium-dependent vascular regulation is perturbed.